1. Field of the Invention.
The present invention relates to a method for making multi-parameter data, and more particularly, concerns a method for making multi-parameter data of dimensionality greater than two viewable by a human analyst on a screen to determine one or more characteristics of cells or other particles of interest.
2. Background Description.
Flow analysis of cells or particles has been relied upon to determine different characteristics of individual particles. Flow cytometry generally refers to those techniques in which cells or other biological particles are caused to flow in a liquid stream, substantially one at a time, so that certain characteristics thereof may be measured, sensed or detected. For example, a liquid sample containing cells is typically directed through a flow cytometry apparatus in a rapidly moving liquid stream so that each cell passes serially, and substantially individually, through a sensing region. As each cell passes through the sensing region, different characteristics of the cell may be determined or detected. If, for example, an incident beam of light is directed into the sensing region, the passing cells scatter such light as they pass therethrough. Further, fluorescence is emitted by autofluorescent or labeled cells which have been excited as a result of passing through the excitation energy of the incident light beam. Light scatter and fluorescence related to these passing cells may be detected to provide various information about the properties or characteristics of those cells.
In addition to emitted or scattered light, other physical or chemical properties of each cell may be detected. For example, light absorbance may be measured as each cell passes through the incident light beam, and electronic cell volume may be measured by using the well-known Coulter principle in which the impedance measurement of a cell passing through an orifice is related to cell volume. These analyses are most useful in areas of research, immunology, hematology and the like. The researcher, for instance, may be interested in determining specific characteristics of individual cells so that the cells may be classified, identified, quantified and perhaps sorted for further investigations or analysis.
Advances in flow cytometry instruments have made the acquisition of multi-parameter data relatively straightforward. Commercially available flow cytometry instruments provide features for the acquisition of simultaneous data including four, five or six different parameters related to each cell under analysis. While some of these data are displayed on a screen to a user in real-time format during the data collection procedures, the data may also be stored in its entirety, in what is called list mode, for further analysis or refinement. With so much information available, multi-parameter or multi-variate analysis of the data may take on a complexity that could render meaningful analysis difficult. Accordingly, different techniques have evolved to make the data handling aspects of flow cytometry analysis not only more meaningful, but also more convenient and simplified for the user.
For example, "gating" techniques have been employed in the visual presentation of data on the CRT screen used in accompaniment to flow cytometers for visualization by the user. In this well-known and utilized technique, only those cells having values for the measured parameters falling within set limits, the gated values, may be seen on the screen. Those cells which fall outside of the gated values are usually eliminated from view so that they vanish from the screen. Of course, this technique simplifies or clarifies the data events displayed on the screen to make it easier for the viewer to study the selected cells of interest. To re-introduce the eliminated cells onto the screen for viewing, extra steps are involved; the user normally will not be able to view the gated events at the same time that the non-gated data events are on the screen.
A more sophisticated version of gating, referred to as window trace integration, was recently described by Kachel, in "Interactive Multi-Window Integration of Two-Parameter Flow Cytometric Data Fields," Cytometry 7:89-92 (1986). In the window trace integration, a cursor point on the screen may be moved by the user over the structures of a two-parameter field leaving a trace on the screen. The window is that field selected by the trace for integration. In the Kachel technique, the trace left by the cursor point is shown with intensified brightness in the ground plane of the two-parameter field. Up to eight independent window traces may be performed on the single two-parameter field, as described by Kachel. In addition to window trace integration, Kachel also describes another technique, referred to as painted field integration, for identifying a field of interest for those cells under analysis in the two-parameter field. In the painted field technique, the cursor point may be used as a brush for "painting" the field so that each channel of the two-parameter field touched by the cursor is marked as part of the integration window. In addition, Kachel describes window trace painting in which the painting may be performed by moving the entire window trace generated by using cursor point painting.
The techniques described by Kachel are indicative of the new approaches for handling two-parameter data for the analysis of cells, particularly with respect to flow cytometric data. The goal is provide the user with the ability to quickly and efficiently discriminate different kinds of cells. However, even though the approach described by Kachel represents an advance in the handling of flow cytometric data, it is limited to a single two dimensional display and does not provide any insight into multi-parameter, multi-dimensional data. It is to a visualization of data of dimensionality greater than two and the achievement of the aforementioned goal of allowing the user to more efficiently and conveniently discriminate cells and cell characteristics, that the present invention is directed.